Development of an Accurate Polymerase Chain Reaction (PCR) Assay for Genetic Sex Identification in Lumpfish (Cyclopterus lumpus) Based on Male-Specific Anti-Mullerian Hormone (amh) Gene

Abstract

The production of lumpfish (Cyclopterus lumpus) has become crucial in controlling sea lice levels in salmonid aquaculture. To improve their breeding, there is a need for early sex identification. The genomic region containing the anti-Müllerian hormone (amh) gene was suggested as the candidate master sex-determining gene in lumpfish. However, the genome of lumpfish contains three copies of amh with ambiguous sex specificity, designated amh1, amh2, and amh3. The study aims to analyse the male-specific region between these amh paralogues for its application as a sex marker. In this study, we utilised polymerase chain reaction (PCR)-based assays to identify the male-specific amh markers in lumpfish and estimate the length of the male-specific region in the lumpfish genome. Our results indicate that a specific genomic region of approximately 27 kilobases (kb), encompassing amh1 and amh2 genes, exhibits male specificity, whereas amh3 is present in both sexes. The developed PCR-based genetic sex identification assays targeting amh1 and amh2 exhibited over 97% concordance with phenotypic records. Further experiments in other members of the Cyclopteridae: Aptocyclus ventricosus, Eumicrotremus taranetzi, and E. asperrimus revealed male-specific amh genome region only in A. ventricosus. Phylogenetic analyses using the available Cyclopteridae amh sequences suggest that male-specific amh arose early in the Cyclopteridae lineage. Our findings, along with the development of the PCR test, hold great promise for the field of lumpfish aquaculture and will also contribute significantly to future investigations aiming to enhance our understanding of the sex-determination system and the evolution of sex chromosomes in teleostean fish.